CN109089882A - A kind of moss tissue culture directly induced by spore and seedling culture method - Google Patents
A kind of moss tissue culture directly induced by spore and seedling culture method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
- A01G24/23—Wood, e.g. wood chips or sawdust
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
A kind of moss tissue culture directly induced by spore and seedling culture method, selection and pre-treatment, spore including sporinite are collected, spore directly sterilize, spore rudiment induction, gemma and protonema shoot proliferation, oscillation differentiation culture, spraying is inoculated with and seedling culture.The method of the present invention improves the efficiency and proliferation efficiency of sterile system foundation, dead low with pollution rate, effectively high into seedling coverage rate after kind of a rate, shoot proliferation rate and transplanting.Compared with prior art, tissue culture reduces by 6.5% into kind of the death rate, and pollution rate reduces by 70.4%, effectively improves 45.4% into kind of rate.Shoot proliferation rate improves 2.63 times, and the moss plant coverage rate that seedling culture is cultivated averagely reaches 85.6%.
Description
Technical field
The invention belongs to plant tissue culture fast breeding technique fields, and in particular in bryophyte tissue-culturing rapid propagation explant selection with
Seedling technology is trained in disinfection, gemma and protonema proliferative induction and field.
Background technique
Moss (bryophytes) is Bryophyta moss guiding principle (Musci) plant, and is distributed the most extensive, kind in the world
Class is most, the simplest higher plant of structure.Bryophytes have to be grown under dim light, high humidity environment, and water content is abundant, life
The special habits such as power is strong, resistance to ridge is thin, rise emphatically in the water and soil conservation of nature, water conservation, Cycle of nutrients and storage
The effect wanted.With the development in city and Garden economy, in recent years in yard beautification, Landscape, moss turf, vertical green
Change, roof garden and application in restoration of the ecosystem and demand increasingly increase.But at present bryophyte artificial propagation there is also
The efficient sterile system of tissue culture is established in more technical problem, especially large-scale production and the bottlenecks such as fast breeding do not solve also completely
Certainly, such as using gametophyte as outside shade when the death rate it is high, when using sporangium as explant because sporangium shell is easy to pollute or capsule cap falls off after
The easy death of spore causes effectively low into kind of rate, and tissue-culturing rapid propagation sterile system difficulty is established, multiplicative stage proliferation rate is high, bottle outlet transplanting
Survive difficulty.Therefore, a large amount of moss materials required in production application mainly or by field largely acquire, or with trophosome
(or gametophyte) and sporinite are that material carries out artificial cutting section propagation, division propagation, spore seed propagation.Excavate meeting in a large amount of fields
Serious to destroy nature and ecological environment, artificial propagation is not only big by environment and seasonal effect, but also dissection, division propagation have increasing
Low efficiency is grown, spore seed propagation has spore again and collects the outstanding problems such as difficult, spore germination rate is low.Therefore, it studies and solves
Moss tissue culture propagation key technology establishes moss reproduction technique efficiently, economic and ecological, too impatient to wait.
Summary of the invention
In order to solve the above technical problems, the present invention is collected by the selection and pre-treatment of sporinite, spore, spore directly disappears
Poison, spore rudiment induction, gemma and protonema shoot proliferation, concussion differentiation culture, spraying inoculation and seedling culture, realize tongue fur
The artificial scale tissue culture propagation of moss and seedling culture.
Technical scheme is as follows:
1. a kind of moss tissue culture directly induced by spore and seedling culture method, which is characterized in that including following step
It is rapid:
(1) selection and pre-treatment of sporinite
When moss sporangium and falx flavescence brown, capsule cap not yet fall off, the sporinite for selecting sporangium full cuts sporangium
Under be put into culture dish, first in the UV lamp cultivate 28-32 minute, then intensity of illumination for 3000lx daylight light under cultivate
2~4 days, 22~28 DEG C of cultivation temperature；
(2) spore is collected
When the capsule cap for the sporangium that step (1) is cultivated starts shedding off, when thering is spore to shed, cuts sporangium or choose out capsule with tweezers
Cap sheds by spore, and collection obtains bryophyte spores；
(3) spore directly sterilizes
The spore that step (2) obtain is placed in centrifuge tube, by spore: the volume ratio of 3~4%w/w hydrogenperoxide steam generator
3~4%w/w hydrogenperoxide steam generator is added for the ratio of 1:10~20, shake 6~after ten minutes, obtain disinfection spore and peroxide
Change the mixed liquor of hydrogen；
(4) spore rudiment induces
Bryophyte spores rudiment induced medium 32-35ml is packed into every culture bottle, with the pipette or dropper by disinfection
The mixed liquor of disinfection spore and hydrogen peroxide that aspiration step (3) obtains is inoculated into the induction of the bryophyte spores rudiment in culture bottle and trains
Primary surface is supported, and gently shaking makes the mixed liquor of the disinfection spore and hydrogen peroxide be uniformly distributed its surface, carries out spore and sprouts
Bud inducement cultivation 30~50 days, spore germination formed gemma and protonema, spore rudiment inducing culturing condition are as follows: temperature 22-28
DEG C, 2500~3000lx of intensity of illumination, initial 1-5 days light application times are 20~24 hours/day, light application time after the 6th day
8 hours/day；The bryophyte spores rudiment induced medium are as follows: improvement 0.5~1.0mg/L+GA of MS+KT30.2mg/L+ agar
6g/L+ white sugar 20g/L, pH value 6.0；
(5) shoot proliferation of gemma and protonema
Solid subculture multiplication medium, the solid subculture multiplication medium that every culture bottle is packed into are packed into new culture bottle
For 32-35ml, be first added in the culture bottle of gemma and protonema that step (4) spore rudiment Fiber differentiation obtains 4~6ml without
Bacterium water forms high concentration gemma and precursor liquid suspension after shake, with aseptic inoculation spoon or oese by high concentration gemma and original
Mycelial suspension is inoculated into solid subculture multiplication medium surface and carries out shoot proliferation culture, after shoot proliferation culture 28-32 days,
Solid subculture multiplication medium surface forms green bacterium colony shape gemma and protonema, every bottle height concentration gemma and precursor liquid suspension
10~15 bottles of inoculation fills the culture bottle of solid subculture multiplication medium, shoot proliferation condition of culture are as follows: and 22~28 DEG C of temperature, light
According to 2500~3000lx of intensity, light application time 8 hours；The solid subculture multiplication medium are as follows: improvement MS+BA0.5~
1.0mg/L+2,4-D 0.2mg/L+IAA 0.3mg/L+ agar 7g/L+ white sugar 20g/L, pH value 6.0；
(6) oscillation differentiation culture
In the culture bottle for foring green bacterium colony shape gemma and protonema after step (5) culture, nothing is added in every bottle
10~15ml of bacterium water is diluted to diluted suspension liquid, and diluted suspension liquid and fluid nutrient medium are pressed diluted suspension liquid: fluid nutrient medium
Volume ratio 1:5 be mixed to get mixed liquor, then by mixed liquor on the shaking table of revolving speed 100r/min shake culture 25~30 days,
Shake culture condition on the shaking table are as follows: 22~28 DEG C of temperature, 2500~3000lx of intensity of illumination, light application time 8 hours, to mixed
It closes when liquid retrogradation and color are bottle green and obtains bottle green suspension；The fluid nutrient medium are as follows: improvement MS+BA0.1mg/L+
NAA 0.2~0.5mg/L+ white sugar 20g/L, pH value 6.0；
(7) spraying inoculation and seedling culture
Moss seedling is trained seedling matrix and is packed into seedlings nursing plate, and the moss seedling training seedling matrix is formulated as follows: will be careless
Charcoal, vermiculite and sawdust are according to turf: vermiculite: sawdust volume ratio is that 6:1:1 ratio mixes；
The bottle green suspension that step (6) culture obtains is diluted with water to obtain dilution, dilution ratio suspends by bottle green
Liquid: volume ratio=1:15~20 of water with the mode that sprays or apply mode of rinsing dilution spray or is applied and rinse moss seedling and train seedling base
Matter surface, and seedlings nursing plate is put on the seedbed in greenhouse progress seedling culture, the top of seedlings nursing plate cover Small plastic shed and in
Clear plastic membrane is covered on Small plastic shed, and the sunshade net that shading rate is 75%, seedling culture item are covered on clear plastic membrane
Part are as follows: from carrying out the 1st day to the 15th day of seedling culture, keep the relative air humidity 90~95% in Small plastic shed, the 16th day
To the 30th day, interval opened the small clear plastic membrane on canopy and ventilates 3-5 times daily, kept the air in Small plastic shed relatively wet
Degree, to seedling culture is completed, opened the small nothing on canopy with after 5 points at night before 10 points of every morning for the 85~90%, the 31st day
Color plastic film is ventilative, keeps the relative air humidity 75~85% in Small plastic shed, from the 1st day to during completing seedling culture,
Canopy temperature is controlled daytime at 20~28 DEG C, night controls the temperature in greenhouse and is not less than 15 DEG C, and moss seedling is kept to train seedling
Stromal surface is wet, completes seedling culture when moss seedling training seedling stromal surface forms moss plant；
It is operated under step (3) to step (6) aseptic condition；
Improvement MS in each culture medium described in step (4), step (5) and step (6) are as follows:
KN03950mg/L, NH4N03825mg/L, MgSO4·7H2O 185mg/L,KH2PO485mg/L, CaCl2·
2H2O220mg/L, MnSO4·4H2O 11.15mg/L, ZnSO4·7H2O 4.3mg/L, H3BO3 3.1mg/L,KI 0.415mg/
L,Na2MoO4·2H2O 0.125mg/L, CuSO4·5H2O 0.0125mg/L, CoCl2·6H2O 0.0125mg/L, Na2-EDTA
18.65mg/L FeSO4·4H2O 13.9mg/L, glycine 2mg/L, Tyiamine Hd element 0.1mg/L, vitamin B6 0.5mg/L,
Niacin 0.5mg/L, inositol 100mg/L, Ca (NO3)2·4H2O 140mg/L。
2. a kind of moss tissue culture directly induced by spore according to technical solution 1 and seedling culture method,
Be characterized in that: repeating step (5), by the switching of the shoot proliferation and subculture of gemma and protonema more than twice realize gemma and
The fast breeding of protonema.
3. a kind of moss tissue culture directly induced by spore and seedling culture method according to technical solution 1 or 2,
It is characterized by: being added 50% by every cubic metre of moss seedling training seedling matrix in the training seedling matrix of the moss seedling described in step (7)
100 grams of carbendazol wettable powder, and be reloaded into seedlings nursing plate after mixing, the subsequent operation is then pressed by dilution
The moss seedling training seedling stromal surface rinsed and be mixed with 50% carbendazol wettable powder in seedlings nursing plate is sprayed or applies, and by seedlings nursing plate
It is put in and carries out the seedling culture on the seedbed in greenhouse.
Compared with prior art, innovative point of the invention and the beneficial effect is that:
1, the efficiency of sterile system foundation is improved.
Capsule cap opening time is not easy to determine when the present invention is directed to field moss sporangium maturation, and it is big directly to collect spore difficulty
The problem of, it takes and first collects the sporangium that sporangium and falx flavescence brown, capsule cap not yet fall off, then ultraviolet light is carried out to sporangium surface
The pre-treatment that fluorescent lamp irradiation culture is carried out after illumination-based disinfection, can be obtained spore more mature, that purity is higher and clean, and adopt
Aggregate measures with 3~4% hydrogenperoxide steam generator directly to spore disinfection, improve the efficiency of sterile system foundation.Firstly,
The spore injured in sporangium is not only not easy to sporangium disinfection using ultraviolet light irradiation, but also can get the sporangium of clean in appearance, then pass through
Daylight light is cultivated under preference temperature and time, obtains mature spore, then directly sterilize to spore using hydrogen peroxide,
Not only small to spore injury, pollution is few, and spore death rate is low, and reaction product is water, and no hazard residue is also not required to use nothing again
The cleaning of bacterium water, can easily and fast establish the sterile tissue culture system of moss.Sporangium is used to carry out mercuric chloride disinfection for explant with existing
It compares, this method tissue culture death rate reduces by 6.5%, and pollution rate reduces by 70.4%, effectively improves 45.4% into kind of rate.[step
(1)、(2)]
2, gemma and protonema use suspension culture in solid subculture multiplication medium, and proliferation efficiency significantly improves.
Moss shoot proliferation material is gemma and precursor liquid suspension [step (5)] in the present invention, it is difficult to as conventional tissue culture
Middle adventitious bud (seedling) subculture equally cuts switching.For this particularity, the gemma and protonema obtain to step (4) adds appropriate nothing
Bacterium water forms high concentration gemma and precursor liquid suspension, then high concentration gemma and protonema inoculation of suspension liquid are increased to solid subculture
Grow media surface culture, a bottle height concentration gemma and precursor liquid suspension mother's bottle is switchable (fills at 10~15 bottles of subculture bottles
The culture bottle of solid subculture multiplication medium), 12.23 times of average proliferation.The height is inoculated in solid subculture multiplication medium
Concentration gemma and precursor liquid suspension can guarantee that gemma and protonema grow required nutrition, moisture, humidity and oxygen demand.
3, the outer seedling culture technique of suitable moss suspension bottle is constructed.
The present invention trains seedling matrix by suitable illumination, humidity stage by stage, temperature control, and suitable moss seedling,
Moss suspension fast-growth after spray (painting) is transplanted forms moss strain and plants, and coverage rate is planted in the moss strain of seedling culture
Average out to 85.6%.It transplants simultaneously and sprays (painting) method after obtaining the dilution of bottle green suspension using concussion differentiation culture, be conducive to be formed
Be evenly distributed and grow neat moss block, can better meet various gardens, greening, household demand, have and good push away
Wide prospect.
In conclusion synergistic effect of the present invention by each step, using gametophyte as outside shade when solving moss tissue culture
When the death rate it is high, because sporangium is easy to pollute or capsule cap falls off, spore death rate height causes effectively into kind of a rate when using sporangium as explant
Low, undergrowth after tissue-culturing rapid propagation sterile system difficulty is established, multiplicative stage proliferation rate is not high and bottle outlet transplanting survives difficult technology
Problem.
The method of the present invention and existing using sporangium as explant, and be trained after plant with protonema differentiation and carry out dissection again
The technology of proliferation is compared, and the tissue culture death rate reduces by 6.5%, and pollution rate reduces by 70.4%, effectively improves 45.4% into kind of rate, subculture
Proliferation rate improves 2.63 times, and the efficiency of moss tissue culture propagation is greatly improved, and the moss plant coverage rate of seedling culture is average
Reach 85.6%, establishes the artificial propagation techniques of suitable batch, large-scale production moss.
Specific embodiment
Below by the embodiment that ' Herba Funariae Hygrometricae ', ' the narrow long beak moss of leaf ' and ' pigeon-wheat ' tissue culture are produced to the present invention do into
The description of one step, following embodiment is conventional method without specified otherwise.
Embodiment 1 (the method for the present invention)
(1) selection and pre-treatment of sporinite
October when the sporangium and falx flavescence brown, capsule cap of ' Herba Funariae Hygrometricae ' not yet fall off, selection physically well develop, sporangium
Full sporinite, sporangium is cut and is put into culture dish, is first cultivated 30 minutes under 20W ultraviolet lamp, then be in intensity of illumination
It is cultivated under the daylight light of 3000lx 3 days, 26 DEG C of cultivation temperature.
(2) spore is collected
When the capsule cap of the sporangium of step (1) culture starts shedding off, when thering is spore to shed, with knife blade incision sporangium or use
Tweezers gently choose out capsule cap and shed by spore, are chosen other sundries such as capsule stalk, sporangium with tweezers after removing, and collection can be used for
The bryophyte spores of tissue cultures；
(3) spore directly sterilizes
The spore that step (2) obtain is placed in centrifuge tube, by spore: the volume ratio of 3%w/w hydrogenperoxide steam generator is 1:
It is 3%w/w hydrogenperoxide steam generator that concentration, which is added, in 15 ratio, after shaking 8 minutes, obtains the mixing of disinfection spore and hydrogen peroxide
Liquid；
(4) spore rudiment induces
Bryophyte spores rudiment induced medium 35ml is packed into every culture bottle.It is inhaled with the pipette or dropper that pass through disinfection
Bryophyte spores rudiment in mixed liquor inoculation (drip in) culture bottle of the disinfection spore and hydrogen peroxide that take step (3) to obtain induces
Media surface, and gently shaking makes the mixed liquor of the disinfection spore and hydrogen peroxide be uniformly distributed its surface, carries out spore
Rudiment Fiber differentiation 35 days, the color for being inoculated into the spore of bryophyte spores rudiment Fiber differentiation primary surface became green from faint yellow
Color, i.e. spore germination form gemma and protonema, spore rudiment inducing culturing condition are as follows: and 25 DEG C of temperature, intensity of illumination 3000lx,
Initially 1-5 days light application times are 24 hours/day, and (intensity of illumination is still for the 6th day later 8 hour/day of light application time
3000lx)；The bryophyte spores rudiment induced medium are as follows: improvement MS+KT 1.0mg/L+GA30.2mg/L+ agar 6g/L+
White sugar 20g/L, pH value 6.0.
(5) shoot proliferation of gemma and protonema
Solid subculture multiplication medium, the solid subculture multiplication medium that every culture bottle is packed into are packed into new culture bottle
For 35ml, 5ml sterile water first is added in the culture bottle of gemma and protonema that step (4) spore rudiment Fiber differentiation obtains,
High concentration gemma and precursor liquid suspension are formed after shake, are hanged high concentration gemma and protonema with aseptic inoculation spoon or oese
Supernatant liquid is inoculated into solid subculture multiplication medium surface and carries out shoot proliferation culture, after shoot proliferation culture 30 days, solid subculture
Multiplying culture primary surface forms green bacterium colony shape gemma and protonema, and every bottle height concentration gemma and protonema inoculation of suspension liquid 10~
15 bottles of culture bottles for filling solid subculture multiplication medium, shoot proliferation rate can reach 10.9~13.4 times.Shoot proliferation culture
Condition are as follows: 25 DEG C of temperature, intensity of illumination 3000lx, light application time 8 hours；The solid subculture multiplication medium are as follows: improvement MS+
BA 0.5mg/L+2,4-D 0.2mg/L+IAA 0.3mg/L+ agar 7g/L+ white sugar 20g/L, pH value 6.0.
The shoot proliferation step (5) for repeating above-mentioned gemma and protonema obtains a large amount of gemma by 3-5 subculture switching
It is proliferated bottle with protonema, average shoot proliferation rate can reach 12.8 times.
(6) oscillation differentiation culture
In the culture bottle for foring green bacterium colony shape gemma and protonema after step (5) culture, nothing is added in every bottle
Bacterium water 12ml is diluted to diluted suspension liquid, and diluted suspension liquid and fluid nutrient medium are pressed diluted suspension liquid: the body of fluid nutrient medium
Product than 1:5 is mixed to get mixed liquor, then by mixed liquor on the shaking table of revolving speed 100r/min shake culture 25 days, the shaking table
Upper shake culture condition are as follows: 25 DEG C of temperature, intensity of illumination 3000lx, light application time 8 hours, the gradually greening of mixed liquor color, to
Mixed liquor retrogradation and color obtain bottle green suspension when being bottle green；The fluid nutrient medium are as follows: improvement MS+BA0.1mg/L+
NAA 0.2mg/L+ white sugar 20g/L, pH value 6.0.
(7) spraying inoculation and seedling culture
Prepare moss seedling and train seedling matrix: by turf, vermiculite and sawdust according to turf: vermiculite: sawdust volume ratio is 6:1:1 ratio
Example mixes moss seedling training seedling matrix.
It is trained in the ratio that 100 grams of 50% carbendazol wettable powder is added in every cubic metre of moss seedling training seedling matrix in moss seedling
Seedling Medium Culture is added 50% carbendazol wettable powder and mixes the moss seedling training for being mixed with 50% carbendazol wettable powder
Matrix is packed into seedlings nursing plate by seedling matrix (hereinafter referred to as: matrix).
The bottle green suspension that step (6) culture obtains is diluted with water to obtain dilution, dilution ratio is outstanding by bottle green
Supernatant liquid: volume ratio=1:15 of water is rinsed mode and dilution is sprayed or applied the matrix rinsed in seedlings nursing plate with the mode that sprays or painting
Surface, and seedlings nursing plate is put in progress seedling culture on the seedbed in greenhouse, Small plastic shed is covered in the top of seedlings nursing plate and in small
Clear plastic membrane is covered on arched shed, covers the sunshade net that shading rate is 75% on clear plastic membrane to keep humidity and half
Negative condition, seedling condition of culture are as follows: the 1st day to the 15th day that seedling culture is carried out on the seedbed in greenhouse is put in from seedlings nursing plate,
By being spaced spraying 3~5 water in Small plastic shed daily, keeping the relative air humidity in Small plastic shed is 90%~95%, the
It 16 days to the 30th day, is ventilated 3-5 times by opening the small clear plastic membrane on canopy daily, keeps the air phase in Small plastic shed
Be 85%~90% to humidity, the 31st day to completing seedling culture, it is small by being opened after 5 points before 10 points of every morning and at night
Ventilative for the clear plastic membrane on canopy, keeping the relative air humidity in Small plastic shed is 75%~85%, by the 45th day after base
Matter surface starts greening, after the 80th day stromal surface formation be evenly distributed, intensive moss plant, when stromal surface forms tongue fur
Seedling culture is completed when moss plant, moss at this time can be used for yard beautification, Landscape, moss turf, vertical green
Change, roof garden and restoration of the ecosystem etc..It is put on the seedbed in greenhouse from seedlings nursing plate and carries out the 1st day of seedling culture to (being formed
Moss plant) complete seedling culture during, control canopy temperature daytime at 20~28 DEG C, night controls the temperature in greenhouse not
Lower than 15 DEG C, and keep stromal surface wet, moss plant coverage rate made of seedling culture reaches 87.5%.
The calculation of moss plant coverage rate:
Moss plant coverage rate (%)=[(have quantity × 1cm of moss2)/gross area cm2] × 100,
The substrate area for rinsing dilution is sprayed or applied as the gross area using step (7), the unit of the gross area is cm2, will be total
Area is drawn as with 1cm2For the grid of a cell, there is 2/3 or more area to overgrow with moss plant in each cell to be
" having moss " less than 1/3 area with moss plant is " no moss " in each cell, counts the quantity of " having moss ",
Moss plant coverage rate can be calculated by above-mentioned formula again.
It is operated under step (3) to step (6) aseptic condition；
Improvement MS in each culture medium described in step (4), step (5) and step (6) are as follows:
KN03950mg/L, NH4N03825mg/L, MgSO4·7H2O 185mg/L,KH2PO485mg/L, CaCl2·
2H2O220mg/L, MnSO4·4H2O 11.15mg/L, ZnSO4·7H2O 4.3mg/L, H3BO3 3.1mg/L,KI 0.415mg/
L,Na2MoO4·2H2O 0.125mg/L, CuSO4·5H2O 0.0125mg/L, CoCl2·6H2O 0.0125mg/L, Na2-EDTA
18.65mg/L FeSO4·4H2O 13.9mg/L, glycine 2mg/L, Tyiamine Hd element 0.1mg/L, vitamin B6 0.5mg/L,
Niacin 0.5mg/L, inositol 100mg/L, Ca (NO3)2·4H2O 140mg/L。
A great number of elements, CaCl in above-mentioned improvement MS2·2H2O, microelement, the half that molysite ingredient is MS culture medium,
Organic element is the full dose of MS culture medium, increases Ca (NO3)2.4H2O 140mg/L。
Embodiment 2 (the method for the present invention)
For embodiment 2 in addition to following steps are different, remaining step is same as Example 1.
(1) selection and pre-treatment of sporinite
In August when the sporangium and falx flavescence brown, capsule cap of ' the narrow long beak moss of leaf ' not yet fall off, it will be developed with scissors good
Good, full sporangium is cut and is put in culture dish, first cultivates 30 minutes under 20W ultraviolet lamp, is then 22 DEG C in temperature, illumination is strong
Degree is daylight light-illuminating 4 days of 2500lx.
(4) spore rudiment induces
Bryophyte spores rudiment induced medium 33ml is packed into every culture bottle.It is inhaled with the pipette or dropper that pass through disinfection
Bryophyte spores rudiment in mixed liquor inoculation (drip in) culture bottle of the disinfection spore and hydrogen peroxide that take step (3) to obtain induces
Media surface, and gently shaking makes the mixed liquor of the disinfection spore and hydrogen peroxide be uniformly distributed its surface, carries out spore
Rudiment Fiber differentiation 48 days, the color for being inoculated into the spore of bryophyte spores rudiment Fiber differentiation primary surface became green from faint yellow
Color, i.e. spore germination form gemma and protonema, spore rudiment inducing culturing condition are as follows: and 22 DEG C of temperature, intensity of illumination 2500lx,
Initially 1-5 days light application times are 20 hours/day, 8 hour/day of light application time after the 6th day；The bryophyte spores rudiment lures
Lead culture medium are as follows: improvement MS+KT 0.6mg/L+GA30.2mg/L+ agar 6g/L+ white sugar 20g/L, pH value 6.0.
(5) shoot proliferation of gemma and protonema
Solid subculture multiplication medium, the solid subculture multiplication medium that every culture bottle is packed into are packed into new culture bottle
For 35ml, 6ml sterile water first is added in the culture bottle of gemma and protonema that step (4) spore rudiment Fiber differentiation obtains,
High concentration gemma and precursor liquid suspension are formed after shake, are hanged high concentration gemma and protonema with aseptic inoculation spoon or oese
Supernatant liquid is inoculated into solid subculture multiplication medium surface and carries out shoot proliferation culture, after shoot proliferation culture 31 days, solid subculture
Multiplying culture primary surface forms green bacterium colony shape gemma and protonema, every bottle height concentration gemma and 15 bottles of protonema inoculation of suspension liquid
Fill the culture bottle of solid subculture multiplication medium；Shoot proliferation condition of culture are as follows: 22 DEG C of temperature, intensity of illumination 2500lx, light
According to the time 8 hours；The solid subculture multiplication medium are as follows: improvement MS+BA 0.8mg/L+2,4-D 0.2mg/L+
IAA0.3mg/L+ agar 7g/L+ white sugar 20g/L, pH value 6.0.
(6) oscillation differentiation culture
In the culture bottle for foring green bacterium colony shape gemma and protonema after step (5) culture, nothing is added in every bottle
Bacterium water 15ml is diluted to diluted suspension liquid, and diluted suspension liquid and fluid nutrient medium are pressed diluted suspension liquid: the body of fluid nutrient medium
Product than 1:5 is mixed to get mixed liquor, then by mixed liquor on the shaking table of revolving speed 100r/min shake culture 25 days, the shaking table
Upper shake culture condition are as follows: 22 DEG C of temperature, intensity of illumination 2500lx, light application time 8 hours, the gradually greening of mixed liquor color, to
Mixed liquor retrogradation and color obtain bottle green suspension when being bottle green；The fluid nutrient medium are as follows: improvement MS+BA0.1mg/L+
NAA 0.5mg/L+ white sugar 20g/L, pH value 6.0.
(7) spraying inoculation and seedling culture
Matrix in seedlings nursing plate is that moss seedling trains seedling matrix.Moss plant coverage rate reaches 83.6%.
Comparative test:
It is that material carries out following tests with ' Herba Funariae Hygrometricae ', ' the narrow long beak moss of leaf ' and ' pigeon-wheat ': 1, different explants and disappears
Malicious method is to the influence into kind of effect；2, influence experimental study of the different subculture methods to proliferation efficiency.It is further by testing
Collected in the verifying present invention by the pre-treatment of sporinite, spore, spore directly sterilize with hydrogen peroxide, spore rudiment induces,
Moss can be improved in the shoot proliferation of gemma and protonema, oscillation differentiation culture, field spraying inoculation and the method for seedling culture
Sterile system is established and the efficiency of shoot proliferation, obtains a large amount of moss tissue-cultured seedling, and moss tissue-cultured seedling is enable well to grow, seedling
Rate is high.
1, different explants and sterilization method are to the influence into kind of effect
It is premenstrual with ' Herba Funariae Hygrometricae ', ' the narrow long beak moss of leaf ' and ' pigeon-wheat ' flavescence brown sporangium and using the method for the present invention respectively
The spore collected after processing is explant, and spore disinfectant with hydrogen peroxide method of the present invention and conventional sporangium mercuric chloride disinfection is respectively adopted
Method carries out explant disinfection.The disinfectant with hydrogen peroxide method is that the hydrogenperoxide steam generator of 3%w/w is directly used explant to sterilize 8
Minute, mercuric chloride sterilization is sterilized 8 minutes to the mercuric chloride solution of explant 0.1%w/w.
Spore is explant: disinfectant with hydrogen peroxide method fills in the centrifuge tube of spore by spore: 3%w/w hydrogen peroxide is molten
It is 3%w/w hydrogenperoxide steam generator that concentration, which is added, in ratio that the volume ratio of liquid is 1:15, after shaking 8 minutes, obtain disinfection spore with
Mixed liquor is inoculated into bryophyte spores rudiment Fiber differentiation primary surface described in embodiment 1 by the mixed liquor of hydrogen peroxide.
Sporangium is explant: mercuric chloride sterilization, and after sporangium is sterilized 8 minutes with the mercuric chloride solution of 0.1%w/w, sporangium is cut
Open, take out spore and be placed in centrifuge tube in spore: the ratio that the volume ratio of sterile water is 1:1.5 is added sterile water and is uniformly mixed shape
At suspension, then by inoculation of suspension liquid to bryophyte spores rudiment Fiber differentiation primary surface described in embodiment 1.
After the above inoculation, [spore rudiment induction training after being cultivated 25 days in the bryophyte spores rudiment induced medium
Support condition it is identical with embodiment 1 step (4)], polluted, death and effectively into the statistics of kind of rate.Pass through the test result of table 1
Show: using the method for the present invention compared with conventional sporangium explant mercuric chloride sterilization, average mortality reduces by 6.5%, pollution
Rate reduces by 70.4%, effectively improves 45.4% into kind of rate.
The different explants of table 1 and sterilization method are to the influence into kind of effect
Remarks: death determination standard is no in bottle after cultivating 25 days or rarely turns green spore.
The death rate (%)=(dead bottle/inoculation bottle) × 100.
Pollution rate (%)=(pollution bottle/inoculation bottle) × 100.
Effectively into kind of a rate (%)=[(inoculation bottle-death bottle-pollution bottle)/inoculation bottle] × 100.
2, the experimental study that different shoot proliferation modes influence proliferation rate
The gemma and protonema that ' Herba Funariae Hygrometricae ', ' the narrow long beak moss of leaf ' and ' pigeon-wheat ' is obtained in step (4) induction, according to
Two kinds of different subculture methods carry out shoot proliferation, and first method is cormus enrichment procedure: first by spore germination induced synthesis
Gemma and protonema be transferred to solid differential medium (solid differential medium formula: improvement MS+BA0.1mg/L+NAA
0.3mg/L+ white sugar 20g/L, pH value 6.0) middle culture 42 days, the plant with cormus is formed, then will be with cormus
The plant segment that plant is cut into 0.1-0.3cm is seeded in culture (every culture in solid subculture multiplication medium described in embodiment 1
Bottle fills solid subculture multiplication medium 35ml, is routinely inoculated in every bottle of culture bottle for filling solid subculture multiplication medium
12-15 plant segment), cormus subculture cycle is 40 days/time；Second method is step of the present invention (5) described formation
High concentration gemma and protonema inoculation of suspension liquid shoot proliferation method in solid subculture multiplication medium described in embodiment 1 are (every
Culture bottle fills solid subculture multiplication medium 35ml, takes the high concentration gemma and precursor liquid suspension with 0.5ml pipette
It drips in the solid subculture multiplication medium, instills high concentration bud in every bottle of culture bottle for filling solid subculture multiplication medium
Spore and precursor liquid suspension 6 drip), subculture cycle is 40 days/time, after culture 80 days, counts the proliferation rate of two kinds of subculture modes.
Influence of the different shoot proliferation modes of table 2 to proliferation rate
Shoot proliferation multiple (again)=subculture bottle number/mother's bottle number.
The test result of table 2 shows: reach 12.23 times using the average proliferation of the method for the present invention, and cormus proliferation
Only 3.37 times, proliferation rate of the present invention improves 2.63 times compared with cormus method of proliferating.
By being tested above as can be seen that can be obtained to three kinds of mosses using the method for the present invention progress tissue cultures
The technical effect effectively into kind of rate and shoot proliferation rate is improved, sterile system is quickly established within a short period of time and quickly carries out
The artificial tissue culture expanding propagation of moss, the method for the present invention are suitable for the batch of different moss types, the production of scale tissue culture.
Claims (3)
1. a kind of moss tissue culture directly induced by spore and seedling culture method, which comprises the following steps:
(1) selection and pre-treatment of sporinite
When moss sporangium and falx flavescence brown, capsule cap not yet fall off, sporangium is cut and is put by the sporinite for selecting sporangium full
Enter in culture dish, first cultivate 28-32 minutes in the UV lamp, then cultivates 2~4 in the case where intensity of illumination is the daylight light of 3000lx
It, 22~28 DEG C of cultivation temperature；
(2) spore is collected
When the capsule cap of sporangium of step (1) culture starts shedding off, when thering is spore to shed, cuts sporangium or choose out capsule cap with tweezers and allow
Spore sheds, and collection obtains bryophyte spores；
(3) spore directly sterilizes
The spore that step (2) obtain is placed in centrifuge tube, by spore: the volume ratio of 3~4%w/w hydrogenperoxide steam generator is 1:
10~20 ratio addition 3~4%w/w hydrogenperoxide steam generator, shake 6~after ten minutes, obtain disinfection spore and hydrogen peroxide
Mixed liquor；
(4) spore rudiment induces
It is packed into bryophyte spores rudiment induced medium 32-35ml in every culture bottle, is drawn with the pipette or dropper that pass through disinfection
The mixed liquor of disinfection spore and hydrogen peroxide that step (3) obtains is inoculated into the bryophyte spores rudiment induced medium in culture bottle
Surface, and gently shaking makes the mixed liquor of the disinfection spore and hydrogen peroxide be uniformly distributed its surface, carries out spore rudiment and lures
Lead culture 30~50 days, spore germination forms gemma and protonema, spore rudiment inducing culturing condition are as follows: 22-28 DEG C of temperature, light
According to 2500~3000lx of intensity, initial 1-5 days light application times are 20~24 hours/day, and light application time 8 is small after the 6th day
When/day；The bryophyte spores rudiment induced medium are as follows: improvement 0.5~1.0mg/L+GA of MS+KT30.2mg/L+ agar 6g/
L+ white sugar 20g/L, pH value 6.0；
(5) shoot proliferation of gemma and protonema
Solid subculture multiplication medium is packed into new culture bottle, the solid subculture multiplication medium that every culture bottle is packed into is
It is sterile that 4~6ml first is added in the culture bottle of gemma and protonema that step (4) spore rudiment Fiber differentiation obtains in 32-35ml
Water forms high concentration gemma and precursor liquid suspension after shake, with aseptic inoculation spoon or oese by high concentration gemma and precursor
Liquid suspension is inoculated into solid subculture multiplication medium surface and carries out shoot proliferation culture, after shoot proliferation culture 28-32 days, Gu
Body subculture multiplication medium surface forms green bacterium colony shape gemma and protonema, and every bottle height concentration gemma and precursor liquid suspension connect
10~15 bottles of kind fills the culture bottle of solid subculture multiplication medium, shoot proliferation condition of culture are as follows: and 22~28 DEG C of temperature, illumination
2500~3000lx of intensity, light application time 8 hours；The solid subculture multiplication medium are as follows: improvement 0.5~1.0mg/ of MS+BA
L+2,4-D 0.2mg/L+IAA 0.3mg/L+ agar 7g/L+ white sugar 20g/L, pH value 6.0；
(6) oscillation differentiation culture
In the culture bottle for foring green bacterium colony shape gemma and protonema after step (5) culture, sterile water is added in every bottle
10~15ml is diluted to diluted suspension liquid, and diluted suspension liquid and fluid nutrient medium are pressed diluted suspension liquid: the body of fluid nutrient medium
Product than 1:5 is mixed to get mixed liquor, then by mixed liquor on the shaking table of revolving speed 100r/min shake culture 25~30 days, it is described
Shake culture condition on shaking table are as follows: 22~28 DEG C of temperature, 2500~3000lx of intensity of illumination, light application time 8 hours, liquid to be mixed
Retrogradation and color obtain bottle green suspension when being bottle green；The fluid nutrient medium are as follows: improvement MS+BA 0.1mg/L+NAA
0.2~0.5mg/L+ white sugar 20g/L, pH value 6.0；
(7) spraying inoculation and seedling culture
Moss seedling is trained seedling matrix and is packed into seedlings nursing plate, and the moss seedling training seedling matrix is formulated as follows: by turf, leech
Stone and sawdust are according to turf: vermiculite: sawdust volume ratio is that 6:1:1 ratio mixes；
The bottle green suspension that step (6) culture obtains is diluted with water to obtain dilution, dilution ratio presses bottle green suspension: water
Volume ratio=1:15~20, with the mode that sprays or apply mode of rinsing dilution sprayed or is applied and rinse moss seedling and train seedling matrix table
Face, and seedlings nursing plate is put in progress seedling culture on the seedbed in greenhouse, Small plastic shed is covered in the top of seedlings nursing plate and in small arch
Clear plastic membrane is covered on canopy, and the sunshade net that shading rate is 75%, seedling condition of culture are covered on clear plastic membrane are as follows:
From carrying out the 1st day to the 15th day of seedling culture, the relative air humidity 90~95% in Small plastic shed is kept, the 16th day to the 30th
It, interval opens the small clear plastic membrane on canopy and ventilates daily, keep the relative air humidity in Small plastic shed be 85~
90%, it to seedling culture is completed, is opened before 10 points of every morning and after at 5 points in evening small thin for the colourless plastics on canopy within the 31st day
Film is ventilative, keeps the relative air humidity 75~85% in Small plastic shed, and from the 1st day to during completing seedling culture, daytime is controlled
At 20~28 DEG C, night controls the temperature in greenhouse and is not less than 15 DEG C canopy temperature, and moss seedling is kept to train seedling stromal surface
It is wet, seedling culture is completed when moss seedling training seedling stromal surface forms moss plant；
It is operated under step (3) to step (6) aseptic condition；
Improvement MS in each culture medium described in step (4), step (5) and step (6) are as follows:
KN03950mg/L, NH4N03825mg/L, MgSO4·7H2O 185mg/L,KH2PO485mg/L, CaCl2·2H2O
220mg/L, MnSO4·4H2O 11.15mg/L, ZnSO4·7H2O 4.3mg/L, H3BO3 3.1mg/L,KI 0.415mg/L,
Na2MoO4·2H2O 0.125mg/L, CuSO4·5H2O 0.0125mg/L, CoCl2·6H2O 0.0125mg/L, Na2-EDTA
18.65mg/L FeSO4·4H2O 13.9mg/L, glycine 2mg/L, Tyiamine Hd element 0.1mg/L, vitamin B6 0.5mg/L,
Niacin 0.5mg/L, inositol 100mg/L, Ca (NO3)2·4H2O 140mg/L。
2. a kind of moss tissue culture directly induced by spore according to claim 1 and seedling culture method, feature
It is: repeats step (5), gemma and precursor is realized by shoot proliferation and the subculture switching of gemma and protonema more than twice
The fast breeding of body.
3. a kind of moss tissue culture directly induced by spore according to claim 1 or 2 and seedling culture method, special
Sign is: the moss seedling described in step (7) is trained in seedling matrix and more than 50% bacterium is added by every cubic metre of moss seedling training seedling matrix
100 grams of clever wettable powder, and be reloaded into seedlings nursing plate after mixing, then dilution is sprayed by the subsequent operation
Or it applies and rinses the moss seedling for being mixed with 50% carbendazol wettable powder in seedlings nursing plate training seedling stromal surface, and seedlings nursing plate is put in
The seedling culture is carried out on seedbed in greenhouse.
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| CN111869566A (en) * | 2020-08-01 | 2020-11-03 | 梁江 | Ginseng free microspore induction culture method |
| CN112335508A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院成都生物研究所 | Application method of moss sporophyte suspension containing chitosan/glucan in bare land greening |
| CN114600771A (en) * | 2022-03-18 | 2022-06-10 | 湖北大学 | Landscape type moss spore large-scale mutagenesis screening method |
| CN116636463A (en) * | 2023-07-10 | 2023-08-25 | 广西壮族自治区中国科学院广西植物研究所 | Cultivation method of sphagnum moss in warm land in high-altitude area |
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| CN111869566A (en) * | 2020-08-01 | 2020-11-03 | 梁江 | Ginseng free microspore induction culture method |
| CN112335508A (en) * | 2020-11-06 | 2021-02-09 | 中国科学院成都生物研究所 | Application method of moss sporophyte suspension containing chitosan/glucan in bare land greening |
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